Terminal Oxidases in Representatives of Different Genera of the Family Microbacteriaceae

The nature of terminal oxidases in representatives of four different genera of the family Microbacteriaceae was studied. It was found that the late-logarithmic and early-stationary cells of all of the investigated strains of the genera Plantibacter and Okibacterium contain the aa ₃-type cytochrome oxidase. Bacteria of the genera Rathayibacter and Agreia synthesize three oxidases, the bb ₃- and aa ₃-type cytochrome oxidases and nonheme cyanide-resistant oxidase, in proportions dependent on the cultivation conditions and the growth phase. Oxygen deficiency in the cultivation medium induces the synthesis of the bd-type oxidase in all of the microorganisms studied. The data obtained provide evidence that the type of terminal oxidases, along with the known chemotaxonomic characteristics, may serve to differentiate the genera of the family Microbacteriaceae at the phenotypic level.     

Optical and resonance Raman spectroscopy of the heme groups of the quinol-oxidizing cytochrome aa3 of Bacillus subtilis.

The cytochrome aa3-type terminal quinol oxidase of Bacillus subtilis catalyzes the four-electron reduction of dioxygen to water. It resembles the aa3-type cytochrome-c oxidase in using heme A as its active-site chromophores but lacks the CuA center and the cytochrome-c oxidizing activity of the mitochondrial enzyme. We have used optical and resonance Raman spectroscopies to study the B. subtilis oxidase in detail. The alpha-band absorption maximum of the reduced minus oxidized enzyme is shifted by 5-7 nm to the blue relative to most other aa3-type oxidases, and accordingly, we designate the Bacillus enzyme as cytochrome aa3-600. The shifted optical spectrum cannot be ascribed to an alteration in the strength of the hydrogen bond between the formyl group of the low-spin heme and its environment, as the Raman line assigned to this mode in aa3-600 has the same frequency and degree of resonance enhancement as the low-spin heme a formyl mode in most other aa3-type oxidases. Raman modes arise at 194 and 214 cm-1 in aa3-600, whereas a single band at about 214 cm-1 is assigned to the iron-histidine stretch for the other aa3-type oxidases. Possible explanations for the occurrence of these two modes are discussed. Comparison of formyl and vinyl modes and heme skeletal vibrational modes in different oxidation states of aa3-600 and of beef heart cytochrome-c oxidase shows a strong similarity, which suggests conservation of essential features of the heme environments in these oxidases.     

Molecular cloning, sequencing, and physiological characterization of the qox operon from Bacillus subtilis encoding the aa3-600 quinol oxidase.

Bacillus subtilis contains two aa3-type terminal oxidases (caa3-605 and aa3-600) catalyzing cytochrome c and quinol oxidation, respectively, with the concomitant reduction of O2 to H2O (Lauraeus, M., Haltia, T., Saraste, M., and Wikstr?m, M. (1991) Eur. J. Biochem. 197, 699-705). Previous studies characterized only the structural genes of caa3-605 oxidase. We isolated the genes coding for the four subunits of a B. subtilis terminal oxidase from a genomic DNA library. These genes, named qoxA to qoxD, are organized in an operon. Examination of the deduced amino acid sequence of Qox subunits showed that this oxidase is structurally related to the large family of mitochondrial-type aa3 terminal oxidases. In particular, the amino acid sequences are very similar to those of subunits of Escherichia coli bo quinol oxidase and B. subtilis caa3-605 cytochrome c oxidase. We produced, by in vitro mutagenesis, a mutation in the qox operon. From the phenotype of the mutant strain devoid of Qox protein, the study of expression of the qox operon in different growth conditions, and the analysis of the deduced amino acid sequence of the subunits, we concluded that Qox protein and aa3-600 quinol oxidase are the same protein. Although several terminal oxidases are found in B. subtilis, Qox oxidase (aa3-600) is predominant during the vegetative growth and its absence leads to important alterations of the phenotype of B. subtilis.     

Terminal oxidases of Bacillus subtilis strain 168: one quinol oxidase, cytochrome aa(3) or cytochrome bd, is required for aerobic growth

The gram-positive endospore-forming bacterium Bacillus subtilis has, under aerobic conditions, a branched respiratory system comprising one quinol oxidase branch and one cytochrome oxidase branch. The system terminates in one of four alternative terminal oxidases. Cytochrome caa(3) is a cytochrome c oxidase, whereas cytochrome bd and cytochrome aa(3) are quinol oxidases. A fourth terminal oxidase, YthAB, is a putative quinol oxidase predicted from DNA sequence analysis. None of the terminal oxidases are, by themselves, essential for growth. However, one quinol oxidase (cytochrome aa(3) or cytochrome bd) is required for aerobic growth of B. subtilis strain 168. Data indicating that cytochrome aa(3) is the major oxidase used by exponentially growing cells in minimal and rich medium are presented. We show that one of the two heme-copper oxidases, cytochrome caa(3) or cytochrome aa(3), is required for efficient sporulation of B. subtilis strain 168 and that deletion of YthAB in a strain lacking cytochrome aa(3) makes the strain sporulation deficient.     

Spectroscopic and genetic evidence for two heme-Cu-containing oxidases in Rhodobacter sphaeroides.

It has recently become evident that many bacterial respiratory oxidases are members of a superfamily that is related to the eukaryotic cytochrome c oxidase. These oxidases catalyze the reduction of oxygen to water at a heme-copper binuclear center. Fourier transform infrared (FTIR) spectroscopy has been used to examine the heme-copper-containing respiratory oxidases of Rhodobacter sphaeroides Ga. This technique monitors the stretching frequency of CO bound at the oxygen binding site and can be used to characterize the oxidases in situ with membrane preparations. Oxidases that have a heme-copper binuclear center are recognizable by FTIR spectroscopy because the bound CO moves from the heme iron to the nearby copper upon photolysis at low temperature, where it exhibits a diagnostic spectrum. The FTIR spectra indicate that the binuclear center of the R. sphaeroides aa3-type cytochrome c oxidase is remarkably similar to that of the bovine mitochondrial oxidase. Upon deletion of the ctaD gene, encoding subunit I of the aa3-type oxidase, substantial cytochrome c oxidase remains in the membranes of aerobically grown R. sphaeroides. This correlates with a second wild-type R. sphaeroides is grown photosynthetically, the chromatophore membranes lack the aa3-type oxidase but have this second heme-copper oxidase. Subunit I of the heme-copper oxidase superfamily contains the binuclear center. Amino acid sequence alignments show that this subunit is structurally very highly conserved among both eukaryotic and prokaryotic species. The polymerase chain reaction was used to show that the chromosome of R. sphaeroides contains at least one other gene that is a homolog of ctaD, the gene encoding subunit I of the aa3-type cytochrome c oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of the ligands of the low spin heme of the cytochrome o ubiquinol oxidase complex using site-directed mutagenesis.

The cytochrome o complex of Escherichia coli is a ubiquinol oxidase which is the predominant respiratory terminal oxidase when the bacteria are grown under high oxygen tension. The amino acid sequences of three of the subunits of this quinol oxidase reveal a substantial relationship to the aa3-type cytochrome c oxidases. The two cytochrome components (b563.5 and o) and the single copper (CuB) present in the E. coli quinol oxidase appear to be equivalent to cytochrome a, cytochrome a3, and CuB of the aa3-type cytochrome c oxidases, respectively. These three prosthetic groups are all located within subunit I of the oxidase. Sequence alignments indicate only six totally conserved histidine residues among all known sequences of subunit I of the cytochrome c oxidases of various species plus the E. coli quinol oxidase. Site-directed mutagenesis has been used to change each of these totally conserved histidines with the presumption that two of these six must ligate to the low spin cytochrome center of the E. coli oxidase. The presence of the low spin cytochrome b563.5 component of the oxidase can be evaluated both by visible absorbance properties and by its EPR spectrum. The results unambiguously indicate that His-106 and His-421 are the ligands of the six-coordinate low spin cytochrome b563.5. Although the data are not definitive in making additional metal ligation assignments of the remaining four totally conserved histidines, a reasonable model is suggested for the structure of the catalytic core of the cytochrome o complex and, by extrapolation, of cytochrome c oxidase.     

Recent studies of the cytochrome o terminal oxidase complex of Escherichia coli

The cytochrome o complex is the predominant terminal oxidase in the aerobic respiratory chain of Escherichia coli when the bacteria are grown under conditions of high aeration. The oxidase is a ubiquinol oxidase and reduces molecular oxygen to water. Electron transport through the enzyme is coupled to the generation of a protonmotive force. The purified cytochrome o complex contains four or five subunits, two protoheme IX (heme b) prosthetic groups, plus at least one Cu. The subunits are all encoded by the cyo operon. Sequence comparisons show that the cytochrome o complex is closely related to the aa3-type cytochrome c oxidase family. Gene fusions have been used to define the topology of each of the gene products. Subunits I, II, III and IV are proposed to have 15, 2, 5 and 3 transmembrane spans, respectively. The fifth gene product (cyoE) encodes a protein with 7 membrane spanning segments, and this may also be a subunit of this enzyme. Fourier transform infrared spectroscopy has been used to monitor CO bound in the active site where oxygen is reduced. These data provide definitive proof that the cytochrome o complex has a heme-copper binuclear center, similar to that present in the aa3-type cytochrome c oxidases. Site-directed mutagenesis is being utilized to define which amino acids are ligands to the heme iron and copper prosthetic groups.     

Temporal expression of Mycobacterium smegmatis respiratory terminal oxidases

Terminal oxidases provide the final step in aerobic respiration by reducing oxygen. The mycobacteria possess two terminal oxidases: a cytochrome c aa3 type and a quinol bd type. We previously isolated a bd-type oxidase knockout mutant of Mycobacterium smegmatis that allowed for functional analysis of the aa3 type without the contribution of bd-type activity. Growth of M. smegmatis LR222 and JAM1 (LR222bd::kan) was monitored and the cytochrome content at different time points examined. No difference in aerobic growth was observed between M. smegmatis LR222 and JAM1. Membranes were obtained from these cultures and the oxidase concentrations were calculated from their spectrum. Although the mutant was producing only one oxidase type, this oxidase did not reach wild-type levels of expression, suggesting an additional mechanism for energizing the membrane. Moreover, the concentration of both oxidases in the wild-type strain dropped when cultures entered stationary phase, which was not the case for the aa3-type oxidase of the mutant strain. This oxidase remained at a constant concentration post mid-log phase. RNase protection assays also demonstrated late growth phase dependent message expression of the bd oxidase and that the subunits I and II genes were cotranscribed as an operon.     

Terminal oxidases in the trypanosomatid Trypanosoma cruzi

Titration of Trypanosoma cruzi respiration with cyanide, with results treated as Dixon plots, indicated the presence of several terminal oxidases. The inhibitions obtained at low cyanide concentrations (0-300 microM), taken together with cyanide effects on cytochrome aa3-deficient, dyskinetoplastic epimastigotes, supported cytochrome aa3 as T. cruzi main terminal oxidase. By increasing cyanide concentration to 1.0 mM, two alternative terminal oxidases could be detected. One of these was active in both kinetoplastic and dyskinetoplastic (cytochrome aa3-deficient) epimastigotes, and azide- and antimycin-insensitive. Complementary cytochrome studies with intact epimastigotes and mitochondrial membranes revealed the presence of cytochromes aa3, b, c558, o and possibly d, as components of the parasite electron transport system. Fractionation studies demonstrated that both o and d were bound to the mitochondrial membrane. Reduction by endogenous substrates and complex formation with cyanide supported cytochrome o as alternative terminal oxidase. EB-cultured, dyskinetoplastic epimastigotes showed the same respiration rate as the kinetoplastic cells, despite the significant decrease of cytochrome aa3, thus indicating adaptive mechanisms that determine the expression of alternative oxidases, whenever the main terminal activity is depressed.     

Ferrous iron oxidation in moderately thermophilic acidophile Sulfobacillus sibiricus N1(T)

The iron-oxidizing system of a moderately thermophilic, extremely acidophilic, gram-positive mixotroph, Sulfobacillus sibiricus N1(T), was studied by spectroscopic, high-performance liquid chromatography and inhibitory analyses. Hemes B, A, and O were detected in membranes of S. sibiricus N1(T). It is proposed that the electron transport chain from Fe2(+) to O? is terminated by 2 physiological oxidases: aa?-type cytochrome, which dominates in the early-exponential phase of growth, and bo?-type cytochrome, whose role in iron oxidation becomes more prominent upon growth of the culture. Both oxidases were sensitive to cyanide and azide. Cytochrome aa? was more sensitive to cyanide and azide, with K(i) values of 4.1 and 2.5 μmol·L?1, respectively, compared with K(i) values for cytochrome bo?, which were 9.5 μmol·L?1 for cyanide and 7.0 μmol·L?1 for azide. This is the first evidence for the participation of a bo?-type oxidase in ferrous iron oxidation. The respiratory chain of the mixotroph contains, in addition to the 2 terminal oxidases, a membrane-bound cytochrome b???.     

Two variants of the assembly factor Surf1 target specific terminal oxidases in Paracoccus denitrificans

Biogenesis of cytochrome c oxidase (COX) relies on a large number of assembly proteins, one of them being Surf1. In humans, the loss of Surf1 function is associated with Leigh syndrome, a fatal neurodegenerative disorder. In the soil bacterium Paracoccus denitrificans, homologous genes specifying Surf1 have been identified and located in two operons of terminal oxidases: surf1q is the last gene of the qox operon (coding for a ba(3)-type ubiquinol oxidase), and surf1c is found at the end of the cta operon (encoding subunits of the aa(3)-type cytochrome c oxidase). We introduced chromosomal single and double deletions for both surf1 genes, leading to significantly reduced oxidase activities in membrane. Our experiments on P. denitrificans surf1 single deletion strains show that both Surf1c and Surf1q are functional and act independently for the aa(3)-type cytochrome c oxidase and the ba(3)-type quinol oxidase, respectively. This is the first direct experimental evidence for the involvement of a Surf1 protein in the assembly of a quinol oxidase. Analyzing the heme content of purified cytochrome c oxidase, we conclude that Surf1, though not indispensable for oxidase assembly, is involved in an early step of cofactor insertion into subunit I.     

Identification of 27-oxo-octacosanoic acid and heptacosane-1,27-dioic acid in Legionella pneumophila

A strain of Escherichia coli having elevated levels of cytochrome bo and lacking the cytochrome bd quinol oxidase was grown in chemostat culture at low copper levels. Such cells had lowered levels of copper and of total cytochrome b. Cytochrome o concentration was unchanged when assayed by conventional CO difference spectroscopy, but apparently diminished by 80% in copper-deficient cells as determined by photodissociation of bound CO at 193 K. This is attributed to depletion of copper in the oxidase of copper-deficient cells, causing rapid recombination of photodissociated CO to haem O. CO recombination was also more sensitive to low intensities of actinic light in copper-depleted oxidase. The results illustrate a further similarity between the active sites of o- and aa3-type terminal oxidases.     

Plasmid-mediated virulence genes in non-typhoid Salmonella serovars

Abstract Among aerobic prokaryotes, many different terminal oxidase complexes have been described. Sequence comparison has revealed that the aa ₃-type cytochrome c oxidase and the bo ₃-type quinol oxidase are variations on the same theme: the heme-copper oxidase. A third member of this family has recently been recognized: the cbb ₃-type cytochrome c oxidase. Here we give an overview, and report that nitric oxide (NO) reductase, a bc -type cytochrome involved in denitrification, shares important features with these terminal oxidases as well. Tentative structural, functional and evolutionary implications are discussed.     

Time-resolved single-turnover of caa(3) oxidase from Thermus thermophilus. Fifth electron of the fully reduced enzyme converts O(H) into E(H) state

The oxidative part of the catalytic cycle of the caa(3)-type cytochrome c oxidase from Thermus thermophilus was followed by time-resolved optical spectroscopy. Rate constants, chemical nature and the spectral properties of the catalytic cycle intermediates (Compounds A, P, F) reproduce generally the features typical for the aa(3)-type oxidases with some distinctive peculiarities caused by the presence of an additional 5-th redox-center-a heme center of the covalently bound cytochrome c. Compound A was formed with significantly smaller yield compared to aa(3) oxidases in general and to ba(3) oxidase from the same organism. Two electrons, equilibrated between three input redox-centers: heme a, Cu(A) and heme c are transferred in a single transition to the binuclear center during reduction of the compound F, converting the binuclear center through the highly reactive O(H) state into the final product of the reaction-E(H) (one-electron reduced) state of the catalytic site. In contrast to previous works on the caa(3)-type enzymes, we concluded that the finally produced E(H) state of caa(3) oxidase is characterized by the localization of the fifth electron in the binuclear center, similar to the O(H)→E(H) transition of the aa(3)-type oxidases. So, the fully-reduced caa(3) oxidase is competent in rapid electron transfer from the input redox-centers into the catalytic heme-copper site.     

A Biochemical Approach to Study the Role of the Terminal Oxidases in Aerobic Respiration in Shewanella oneidensis MR-1

The genome of the facultative anaerobic γ-proteobacterium Shewanella oneidensis MR-1 encodes for three terminal oxidases: a bd-type quinol oxidase and two heme-copper oxidases, a A-type cytochrome c oxidase and a cbb ₃-type oxidase. In this study, we used a biochemical approach and directly measured oxidase activities coupled to mass-spectrometry analysis to investigate the physiological role of the three terminal oxidases under aerobic and microaerobic conditions. Our data revealed that the cbb ₃-type oxidase is the major terminal oxidase under aerobic conditions while both cbb ₃-type and bd-type oxidases are involved in respiration at low-O₂ tensions. On the contrary, the low O₂-affinity A-type cytochrome c oxidase was not detected in our experimental conditions even under aerobic conditions and would therefore not be required for aerobic respiration in S. oneidensis MR-1. In addition, the deduced amino acid sequence suggests that the A-type cytochrome c oxidase is a ccaa ₃-type oxidase since an uncommon extra-C terminal domain contains two c-type heme binding motifs. The particularity of the aerobic respiratory pathway and the physiological implication of the presence of a ccaa ₃-type oxidase in S. oneidensis MR-1 are discussed.     

Sulfite oxidation catalyzed by aa(3)-type cytochrome c oxidase in Acidithiobacillus ferrooxidans

Sulfite is produced as a toxic intermediate during Acidithiobacillus ferrooxidans sulfur oxidation. A. ferrooxidans D3-2, which posseses the highest copper bioleaching activity, is more resistant to sulfite than other A. ferrooxidans strains, including ATCC 23270. When sulfite oxidase was purified homogeneously from strain D3-2, the oxidized and reduced forms of the purified sulfite oxidase absorption spectra corresponded to those of A. ferrooxidans aa(3)-type cytochrome c oxidase. The confirmed molecular weights of the α-subunit (52.5 kDa), the β-subunit (25 kDa), and the γ-subunit (20 kDa) of the purified sulfite oxidase and the N-terminal amino acid sequences of the γ-subunit of sulfite oxidase (AAKKG) corresponded to those of A. ferrooxidans ATCC 23270 cytochrome c oxidase. The sulfite oxidase activities of the iron- and sulfur-grown A. ferrooxidans D3-2 were much higher than those cytochrome c oxidases purified from A. ferrooxidans strains ATCC 23270, MON-1 and AP19-3. The activities of sulfite oxidase purified from iron- and sulfur-grown strain D3-2 were completely inhibited by an antibody raised against a purified A. ferrooxidans MON-1 aa(3)-type cytochrome c oxidase. This is the first report to indicate that aa(3)-type cytochrome c oxidase catalyzed sulfite oxidation in A. ferrooxidans.     

Bacillus subtilis expresses two kinds of haem-A-containing terminal oxidases

The expression of two different aa3-type cytochrome oxidases is demonstrated in Bacillus subtilis. One of them (denoted caa3-605), was predicted by DNA-sequencing of Bacillus cytochrome oxidase genes, but has not been found previously. It contains covalently bound haem C in subunit II and is very similar to the enzyme previously described in the thermophilic bacterium PS3. The other oxidase (denoted aa3-600) deviates from most known oxidases of aa3 type, and is probably identical with the oxidase described by de Vrij et al. [de Vrij, W., Azzi, A. & Konings, W. N. (1983) Eur. J. Biochem. 131, 97-103]. It shows no immunological cross-reactivity to the PS3 enzyme and differs from this spectroscopically; it contains no CuA and does not oxidise cytochrome c despite of its haem-A chromophores. It catalyses oxidation of quinols, which is proposed to be its physiological function.     

Over-expression of cbaAB genes of Bacillus stearothermophilus produces a two-subunit SoxB-type cytochrome c oxidase with proton pumping activity

We constructed expression plasmids containing cbaAB, the structural genes for the two-subunit cytochrome bo(3)-type cytochrome c oxidase (SoxB type) recently isolated from a Gram-positive thermophile Bacillus stearothermophilus. B. stearothermophilus cells transformed with the plasmids over-expressed an enzymatically active bo(3)-type cytochrome c oxidase protein composed of the two subunits, while the transformed Escherichia coli cells produced an inactive protein composed of subunit I without subunit II. The oxidase over-expressed in B. stearothermophilus was solubilized and purified. The oxidase contained protoheme IX and heme O, as the main low-spin heme and the high-spin heme, respectively. Analysis of the substrate specificity indicated that the high-affinity site is very specific for cytochrome c-551, a cytochrome c that is a membrane-bound lipoprotein of thermophilic Bacillus. The purified enzyme reconstituted into liposomal vesicles with cytochrome c-551 showed H(+) pumping activity, although the efficiency was lower than those of cytochrome aa(3)-type oxidases belonging to the SoxM-type.     

A cytochrome bb'-type quinol oxidase in Bacillus subtilis strain 168.

The aerobic respiratory system of Bacillus subtilis 168 is known to contain three terminal oxidases: cytochrome caa(3), which is a cytochrome c oxidase, and cytochrome aa(3) and bd, which are quinol oxidases. The presence of a possible fourth oxidase in the bacterium was investigated using a constructed mutant, LUH27, that lacks the aa(3) and caa(3) terminal oxidases and is also deficient in succinate:menaquinone oxidoreductase. The cytochrome bd content of LUH27 can be varied by using different growth conditions. LUH27 membranes virtually devoid of cytochrome bd respired with NADH or exogenous quinol as actively as preparations containing 0.4 nmol of cytochrome bd/mg of protein but were more sensitive to cyanide and aurachin D. The reduced minus oxidized difference spectra of the bd-deficient membranes as well as absorption changes induced by CO and cyanide indicated the presence of a "cytochrome o"-like component; however, the membranes did not contain heme O. The results provide strong evidence for the presence of a terminal oxidase of the bb' type in B. subtilis. The enzyme does not pump protons and combines with CO much faster than typical heme-copper oxidases; in these respects, it resembles a cytochrome bd rather than members of the heme-copper oxidase superfamily. The genome sequence of B. subtilis 168 contains gene clusters for four respiratory oxidases. Two of these clusters, cta and qox, are deleted in LUH27. The remaining two, cydAB and ythAB, encode the identified cytochrome bd and a putative second cytochrome bd, respectively. Deletion of ythAB in strain LUH27 or the presence of the yth genes on plasmid did not affect the expression of the bb' oxidase. It is concluded that the novel bb'-type oxidase probably is cytochrome bd encoded by the cyd locus but with heme D being substituted by high spin heme B at the oxygen reactive site, i.e. cytochrome b(558)b(595)b'.     

The sequence of the cyo operon indicates substantial structural similarities between the cytochrome o ubiquinol oxidase of Escherichia coli and the aa3-type family of cytochrome c oxidases

The cytochrome o complex is one of two ubiquinol oxidases in the aerobic respiratory system of Escherichia coli. This enzyme catalyzes the two-electron oxidation of ubiquinol-8 which is located in the cytoplasmic membrane, and the four-electron reduction of molecular oxygen to water. The purified oxidase contains at least four subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and has been shown to couple electron flux to the generation of a proton motive force across the membrane. In this paper, the DNA sequence of the cyo operon, containing the structural genes for the oxidase, is reported. This operon is shown to encode five open reading frames, cyoABCDE. The gene products of three of these, cyoA, cyoB, and cyoC, are clearly related to subunits II, I, and III, respectively, of the eukaryotic and prokaryotic aa3-type cytochrome c oxidases. This family of cytochrome c oxidases contain heme a and copper as prosthetic groups, whereas the E. coli enzyme contains heme b (protoheme IX) and copper. The most striking sequence similarities relate the large subunits (I) of both the E. coli quinol oxidase and the cytochrome c oxidases. It is likely that the sequence similarities reflect a common molecular architecture of the two heme binding sites and of a copper binding site in these enzymes. In addition, the cyoE open reading frame is closely related to a gene denoted ORF1 from Paracoccus dentrificans which is located in between the genes encoding subunits II and III of the cytochrome c oxidase of this organism. The function of the ORF1 gene product is not known. These sequence relationships define a superfamily of membrane-bound respiratory oxidases which share structural features but which have different functions. The E. coli cytochrome o complex oxidizes ubiquinol but has no ability to catalyze the oxidation of reduced cytochrome c. Nevertheless, it is clear that the E. coli oxidase and the aa3-type cytochrome c oxidases must have very similar structures, at least in the vicinity of the catalytic centers, and they are very likely to have similar mechanisms for bioenergetic coupling (proton pumping).